Missios, Pavlos

Abstract Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of B-cell Non-Hodgkin Lymphoma (NHL), representing a third of all new cases. DLBCL is further sub-divided into various molecular sub-types based on gene expression and co-occurring genetic alterations. Gene expression based- subtypes include the germinal center B-cell (GCB) and the activated B-cell type (ABC) subtypes, with the ABC sub-type having a poorer prognosis than the GCB sub-type. Interestingly, 30-40% of all DLBCL patients harbor mutations in key epigenetic regulators, EZH2, KMT2D, CREBBP, EP300, and mutations in histone proteins themselves. Through an unbiased cell-based phenotypic screen, we discovered that inhibition of lysine demethylases, specifically KDM4C and KDM4A, represents a vulnerability across 15 different DLBCL cell lines including germinal center B-cell (GCB) and activated B-cell (ABC) type lines, with a GI50 value between 75nM to ~200nM, while sparing leukemia lines. Consistently, treatment of xenograft-based animal models of DLBCL with a low dose of KDM4A/KDM4C inhibitor delivered intra-peritoneally three times a week, results in a drastic reduction of tumor burden. Both KDM4A and KDM4C catalyze the removal of histone H3 K9 di- and tri-methylation (H3K9me2/3) and H3K36 di- and tri-methylation (H3K36me2/3). H3K9me2/3 is associated with promoter and enhancer repression, while H3K36me2/3 is present in gene bodies during transcription but also functions as a chromatin repressor Consistently, we have identified key enhancers, including those associated with IKZF1, that are "decommissioned" after inhibition of KDM4A/KDM4C. Repressed enhancer activity, through loss of H3K27 acetylation and H3K4me1, and gain of H3K9me2/3, results in a rapid transcriptional downregulation of IKZF1 and its partners including IKZF3. Given the role of IKZF1 in the transcriptional regulation of key B Cell Receptor (BCR) signaling components, we show that KDM4A/KDM4C inhibition leads to a downregulation of SYK, a proximal BCR-signaling component, which likely precedes DLBCL cell apoptosis. In addition, we observed an activation of extra-lineage transcription factors such as CEBPA and CEBPB, which are normally repressed by IKZF1 in the lymphoid lineage. A concomitant downregulation of the B-cell gene expression program and an upregulation of the myeloid (CD14+ monocytic) gene expression program is also observed, implying a "trans-differentiation" of DLBCL cells into the monocyte lineage. This lineage-switch correlates with an increased population of CD14+ expressing cells. Finally, using DLBCL patient data sets, we can show that over-expression of either KDM4A or KDM4C is associated with poor prognosis in DLBCL patients. In summary, we have discovered that KDM4A/KDM4C inhibition results in an increase of repressive histone modifications at several intra-genic "enhancers" of genes that are responsible for the survival and proliferation of DLBCL cells. The elucidation of this unique epigenetic mechanism provides a strong rationale for the development of novel targeted therapies against both multiple subtypes of DLBCL. Figure. Figure. Disclosures Shipp: Bayer: Research Funding; AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract LIN28 is well known as a RNA-binding protein and a suppressor of microRNA biogenesis, by selectively blocking the processing of let-7 precursors. It plays diverse functions in cellular reprogramming, development, metabolism and tumorigenesis. Many of these functions are executed through its ability to inhibit let-7 maturation. However, little is known about its function independent of let-7. Here we made zebrafish transgenic lines expressing high levels of either wild-type or mutant LIN28B (does not block the maturation of let-7) in the peripheral sympathetic nervous system driven by the dopamine beta-hydroxylase promoter. We bred these lines with a transgenic zebrafish line overexpressing the MYCN oncogene in these cells. Either wild-type or mutant LIN28B overexpression accelerated the onset and increased the penetrance of MYCN-induced neuroblastoma, despite the fact that only wild-type LIN28B blocked let-7 maturation compared to MYCN-only tumors. Mechanistically, both wild-type and mutant LIN28B overexpression enhanced MYCN-induced hyperplasia by increasing cell proliferation in the sympathoadrenal lineage. Further studies revealed that overexpression of either wild-type or mutant LIN28B resulted in hyperphosphorylation of AKT on both Ser473 and Thr308 in zebrafish tumors and also serum-deprived human neuroblastoma cell lines. Both wild-type and mutant LIN28B interacted with IGF2BP1, a known interactor that increases stability and translation of a subset of RNAs. We found IGF2 RNA levels to be increased in response to either form of LIN28B. Coincident with IGF2 overexpression, IGF1R was phosphorylated, providing an explanation for PI3K-AKT activation in LIN28B overexpressing cells. Finally, overexpression of a constitutively active, myristoylated murine Akt2 (myr-mAkt2) alone in zebrafish induced ganglioneuroma in the interregnal gland (the zebrafish equivalent of the human adrenal medulla), without a requirement for MCYN overexpression. Thus, our studies indicate that overexpression of LIN28B leads to AKT activation mediated through its interaction with IGF2BP1 and subsequent upregulation of IGF2. This pathway provides a mechanism underlying enhanced transformation in LIN28B overexpressing neuroblastomas, which is independent of the inhibitory activity of LIN28B on let-7 maturation. Citation Format: Ting Tao, John T. Powers, Hui Shi, Pavlos Missios, Antonio R. Perez-Atayde, Shizhen Zhu, George Q. Daley, Thomas A. Look. LIN28B-mediated let-7 independent activation of AKT promotes neuroblastoma pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1040. doi:10.1158/1538-7445.AM2017-1040